JP5046926B2 - Inflammatory disease preventive or therapeutic agent - Google Patents

Description

  The present invention relates to a prophylactic or therapeutic agent for inflammatory diseases comprising 6,9,12,15-hexadecatetraenoic acid or a pharmaceutically acceptable salt or ester thereof as an active ingredient, the prevention or treatment of inflammatory diseases. The present invention relates to a method for preventing or treating inflammatory diseases, wherein the use thereof for producing a medicament for treatment, or a pharmacologically effective amount thereof is administered to a human or non-human warm-blooded animal.

Inflammation is (1) a phenomenon resulting from the body's defense reaction against pathogenic microorganisms (2) classically accompanied by redness, pain, swelling, and fever (3) accompanied by loss of tissue function (4) secreted from immune system cells Cytokines mediate various responses. The collective term for diseases associated with these conditions is inflammatory disease. Cyclooxygenase and various cytokines play a central role as factors involved. In many cases, cyclooxygenase inhibitors and steroids are effective for inflammatory diseases. However, in the case of chronic inflammatory diseases, currently available drugs are limited due to problems such as side effects caused by long-term administration. In particular, chronic inflammatory diseases such as rheumatism, Crohn's disease, ulcerative colitis, asthma, atopic dermatitis, psoriasis, systemic lupus erythematosus, gout and the like are recognized as typical chronic inflammatory diseases.
Crohn's disease and ulcerative colitis are diseases characterized by chronic inflammation of various parts of the digestive tract. In both diseases, sulfasalazine drugs are often effective, but no cure is known. In recent years, due to the development of therapeutic methods, as a potent immunoregulatory therapy, interleukin-1 blocker, antibody against interleukin-12, and the most effective monoclonal antibody against tumor necrosis factor (THF-alpha) are clinically applied. In these diseases, certain cytokines (TNF-alpha, IL-1, etc.) are suppressed more than general immunosuppressive drugs such as cyclosporine and azathioprine, which are feared to have side effects. Expectations are rising for therapy.
Similarly, in the treatment of rheumatism, TNF-alpha antibody drugs such as infliximab have been approved, and the significance of suppressing such cytokines has been recognized.

  Polyunsaturated fatty acids are the names for unsaturated fatty acids that generally have multiple double bonds, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which are abundant in fish oil. To do. Polyunsaturated fatty acids are roughly classified into n-3 series such as α-linolenic acid, EPA, DHA and n-6 series such as linoleic acid, γ-linolenic acid, arachidonic acid and the like. They are classified from the viewpoint that the position of the double bond is present at the third position from the methyl terminus and that at the sixth position. Many n-6 series fatty acids are contained in plants, while n-3 series fatty acids are contained in aquatic organisms. Recently, n-3 and n-6 fatty acids are ingested in a balanced manner. It is important to do. Fifth revised Japanese nutritional requirements In 1994, it was reported that n-6: n-3 = 4: 1 balance was good. Some scholars need to raise this balance to 1: 1.

  Polyunsaturated fatty acids, particularly fatty acids belonging to n-3 and n-6 systems, have various physiological functions, and research has been actively conducted in various fields. The idea that the intake balance of n-3 or n-6 fatty acids contained in a meal is important for inflammation, immunity, or allergy, which is the main focus of the present invention, is widely recognized. Arachidonic acid plays a leading role in the so-called arachidonic acid cascade, and as a raw material for arachidonic acid metabolites, it is involved in the worsening of symptoms such as inflammation and allergies. The α-3 linolenic acid, EPA and DHA, which are n-3 fatty acids, weaken the action of arachidonic acid (specifically, it inhibits the release of arachidonic acid from the cell membrane by cPLA2; for receptors on which arachidonic acid metabolites act. Thus, it is widely known that it has an action of suppressing inflammation by an action mechanism. In addition, n-3 fatty acids such as hexadecatetraenoic acid, octadecatetraenoic acid, and octadecatrienoic acid have been reported to have anti-inflammatory / antiallergic effects. As described above, many of the n-3 fatty acids exhibit an anti-inflammatory / anti-allergic action and are known to have clearly different actions from the n-6 fatty acids.

  On the other hand, highly unsaturated fatty acids having a double bond in the carbon at the methyl terminal, that is, n-1 fatty acids are extremely rare fatty acids. 6,9,12,15-Hexadecatetraenoic acid (also referred to as C16: 4 (n-1)) is a kind of highly unsaturated fatty acid characteristically contained in sardines and the like. Non-Patent Document 1 reports on the isolation and analysis method. There are few reports on the study of the physiological activity related to n-1 fatty acids, not limited to 6,9,12,15-hexadecatetraenoic acid.

This 6,9,12,15-hexadecatetraenoic acid isomer, 4,7,10,13-hexadecatetraenoic acid, is an n-3 fatty acid with various known actions. Therefore, in the report on the action of the mixture of n-3 fatty acids, there is a report as one component in the mixture of n-3 fatty acids (Non-patent Document 2 etc.), but a purified single substance 4,7,10,13-hexadecatetraenoic acid derived from seaweed is leukotriene B ₄ , leukotriene C ₄ , 5-hydroxyeicosa in mouse mast cell (MC / 9). There are reports that the production of tetraenoic acid is suppressed (Non-patent Document 3) and reports that seaweed-derived 4,7,10,13-hexadecatetraenoic acid has a cell activation effect (Patent Document 1). .

JP-A-2005-23028 Journal of the American chemists’Society, 66 (9), 1323-1325, 1989. Biochimica et Biophysica Acta, 1095, 187-195, 1991. Biosci. Biotechnol. Biochem., 62 (7), 1412-1415, 1998.

  It is an object of the present invention to provide a therapeutic agent for inflammatory diseases having anti-inflammatory action, anti-allergic action, and immunosuppressive action.

As a result of intensive studies on the pharmacological action of 6,9,12,15-hexadecatetraenoic acid, the present inventors have found that the compound has an inhibitory action on the production of interleukin, TNF-α and the like. Completed the invention.
The gist of the present invention is a prophylactic or therapeutic agent for inflammatory diseases comprising 6,9,12,15-hexadecatetraenoic acid or a pharmaceutically acceptable salt or ester thereof as an active ingredient.
The gist of the present invention is a kit for the prevention or treatment of inflammatory diseases comprising the above-mentioned therapeutic agent for inflammatory diseases and instructions for use.
The gist of the present invention is the use of 6,9,12,15-hexadecatetraenoic acid or a pharmaceutically acceptable salt or ester thereof for producing a prophylactic or therapeutic agent for inflammatory diseases.
The present invention provides an effective amount of 6,9,12,15-hexadecatetraenoic acid or a pharmaceutically acceptable salt or ester thereof for human or non-human warm blood in need of prevention or treatment of inflammatory diseases. The gist is a method for preventing or treating inflammatory diseases including administration to animals.
The gist of the present invention is a functional food for the prevention or treatment of inflammatory diseases containing 6,9,12,15-hexadecatetraenoic acid or a salt or ester acceptable as a food additive thereof as an active ingredient.
The gist of the present invention is a functional food for preventing or treating inflammatory diseases including the functional food for treating inflammatory diseases and instructions for use for inflammatory diseases.
As a pharmaceutically acceptable salt of 6,9,12,15-hexadecatetraenoic acid in the above-mentioned preventive or therapeutic agent for inflammatory diseases, use, prevention or treatment method, functional food for prevention or treatment, Sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, pyridine salt and triethylamine salt are preferable, and sodium salt is particularly preferable. In addition, as pharmaceutically acceptable esters of 6,9,12,15-hexadecatetraenoic acid, triglycerides, diglycerides containing 6,9,12,15-hexadecatetraenoic acid as a constituent fatty acid, Or a monoglyceride or a lower alcohol ester of 6,9,12,15-hexadecatetraenoic acid is preferred. The present invention is effective for chronic inflammatory diseases among inflammatory diseases.

  The 6,9,12,15-hexadecatetraenoic acid of the present invention, or a pharmaceutically acceptable salt or ester thereof, suppresses the production of various cytokines, and is an anti-inflammatory agent / anti-allergic agent / immunosuppressive agent It has an effective prophylactic or therapeutic effect against various inflammatory diseases involving inflammatory cells such as

The 6,9,12,15-hexadecatetraenoic acid used in the present invention is a straight chain fatty acid having 16 carbon atoms and has double bonds at the 6th, 9th, 12th and 15th positions. Sometimes written as C16: 4.
The pharmaceutically acceptable salt or ester of 6,9,12,15-hexadecatetraenoic acid of the present invention can be produced by the following method. Generally, fish oil used as a raw material for 6,9,12,15-hexadecatetraenoic acid is mostly triglyceride (TG), while 6,9,12,15-hexadecatetraenoic acid is an acyl residue. It exists as a group. Fish oil also contains TGs with high melting point fatty acids such as palmitic acid and stearic acid. By cooling and crystallizing and removing these high melting point components, 6,9,12, 15-hexadecatetraenoic acid can be concentrated. This method is called low temperature fractionation or wintering. In the case of fish oil, a method of dissolving oil in a solvent such as acetone, cooling, precipitating crystals, and separating is generally performed. However, since 3 molecules of fatty acid are bonded to TG, it is difficult to concentrate 6,9,12,15-hexadecatetraenoic acid to a certain level or more with TG. When highly concentrated, concentrate TG in the form of free fatty acids or monoesters. In order to separate a specific fatty acid from the form of free fatty acid or monoester, methods such as distillation, urea addition, column chromatography, enzyme reaction, supercritical fluid extraction, silver complex formation, low-temperature fractionation, and solvent fractionation are used. Fish oil contains a very wide variety of constituent fatty acids, and highly purified by a single purification method is difficult, and a plurality of methods are used in combination.

In the present invention, the pharmaceutically acceptable salt of 6,9,12,15-hexadecatetraenoic acid is preferably a pharmaceutically or physiologically acceptable alkali addition salt. For example, a salt with sodium, potassium, magnesium, calcium, ammonium, pyridine, triethylamine, or the like is used.
In the present invention, the pharmaceutically acceptable ester of 6,9,12,15-hexadecatetraenoic acid includes a triglyceride containing 6,9,12,15-hexadecatetraenoic acid as a constituent fatty acid, Diglycerides, or monoglycerides, or lower alcohol esters of 6,9,12,15-hexadecatetraenoic acid are preferred. As long as it contains 6,9,12,15-hexadecatetraenoic acid as a constituent fatty acid of triglyceride and diglyceride, it may contain another fatty acid as a constituent fatty acid. For example, when triglyceride derived from fish oil is used as a raw material, various fatty acids having 14 to 22 carbon atoms and 0 to 6 double bonds are included as constituent fatty acids, but 6,9,12,15-hexadecatetraenoic acid is What is necessary is just to concentrate to the density | concentration which can take in required amount. As the lower alcohol ester, methanol and ethanol ester are preferable.

  The 6,9,12,15-hexadecatetraenoic acid, or a pharmaceutically acceptable salt or ester thereof used in the present invention is administered in various forms. The administration form is not particularly limited, and is determined according to various preparation forms, patient age, sex and other conditions, the degree of disease, and the like. For example, in the case of tablets, pills, powders, granules, syrups, solutions, suspensions, emulsions, granules and capsules, they are orally administered. In the case of injections, they are administered alone or mixed with normal replacement fluids such as glucose and amino acids, and administered intravenously, and if necessary, administered alone intramuscularly, intradermally, subcutaneously, or intraperitoneally. Is done. In the case of a suppository, it is administered intrarectally. Oral administration is preferred. Since 6,9,12,15-hexadecatetraenoic acid is a liquid oil that is easily oxidized, it needs to be subjected to an anti-oxidation treatment such as filling in capsules or adding an antioxidant.

  These various preparations are prepared in accordance with conventional methods such as excipients, binders, disintegrants, lubricants, solubilizers, flavoring agents, flavoring agents, coating agents, sweeteners, coloring agents, lubricants, stabilizers, It can be formulated using known adjuvants that can be generally used in the field of pharmaceutical preparations such as preservatives and preservatives. In molding into tablets, conventionally known carriers can be widely used as carriers, such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and the like. Excipient, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone sugar binder, dried starch, sodium alginate, agar powder, laminaran Powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose and other disintegrants, sucrose, stearin, cacao butter, hydrogenated oil and other disintegration inhibitors, 4th grade Ammoni Absorption accelerators such as mud base, sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearate, boric acid powder, polyethylene glycol, etc. Examples of these lubricants can be given. Furthermore, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

In molding into the form of a pill, those conventionally known in this field can be widely used as a carrier. For example, glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc and other excipients, gum arabic powder Examples thereof include binders such as tragacanth powder, gelatin and ethanol, and disintegrants such as lamina lankanten.
In the case of forming into a suppository, conventionally known carriers can be widely used as carriers, such as polyethylene glycol, cacao butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like. Can do.
When prepared as injections, the solutions and suspensions are preferably sterilized and isotonic with blood. In the case of molding into these solutions, emulsions and suspensions, these solutions are used as diluents. Any of those commonly used in the field can be used, and examples thereof include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of sodium chloride, glucose, or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a normal solubilizer, buffer, soothing agent, etc. May be added. Furthermore, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, and other pharmaceuticals as needed.

  The amount of the active ingredient compound contained in the above pharmaceutical preparation is not particularly limited and is appropriately selected over a wide range. When filling a capsule, it is filled with liquid 6,9,12,15-hexadecatetraenoic acid only. In the case of preparing other preparations, it is usually appropriate to contain 1 to 70% by weight in the total composition. In the present invention, the dose of 6,9,12,15-hexadecatetraenoic acid, or a pharmaceutically acceptable salt or ester thereof varies depending on symptoms, age, administration method, etc. In some cases, it is desirable to administer 1-1200 mg per day, preferably 50-1000 mg per day, in one or several divided doses depending on the symptoms. In the case of intravenous administration, it is desirable to administer 0.01 to 500 mg, preferably 0.1 to 200 mg per day for adults in one or several divided doses according to the symptoms.

  Functional foods containing 6,9,12,15-hexadecatetraenoic acid may be in the form of capsules or tablets, or added to beverages such as milk, soy milk, soft drinks, or foods But you can.

The prophylactic or therapeutic agent for inflammatory diseases of the present invention is effective for various inflammatory diseases because it has anti-inflammatory, anti-allergic, and immunosuppressive effects due to mechanisms such as TNF-α production inhibition and cytokine production inhibition. is there. Particularly suitable for chronic inflammatory diseases. Examples include ulcerative colitis, inflammatory bowel diseases such as Crohn's disease, arthritis such as rheumatism, and the like.
6,9,12,15-Hexadecatetraenoic acid is a fatty acid contained in fishes such as sardines, herrings, menhadens, etc. that have been ingested by humans for many years as a dietary habit (Reference: RG Ackman: Fatty Acid Composition of Fish Oils, In Nutritional Evaluation of Long-Chain Fatty Acid in Fish Oil (Edited by SM Barlow & ME Stansby: Academic Press, London, New York, (1982) pp.25-88). Literature; RG Ackman, CA Eaton & J. Hingley: Fillet Fat and Fatty Acid Details for Newfoundland Winter Herring, Canadian Institute of Food Science and Technology Journal, 8 (3), 155-159 (1975)).

  Examples of the present invention will be described below, but the present invention is not limited thereto.

Activity evaluation of 6,9,12,15-hexadecatetraenoic acid using an in vitro cultured cell system
The inhibitory effect of 6,9,12,15-hexadecatetraenoic acid (C16: 4) on the production of various cytokines was evaluated in an assay system for cultured cells. In the test of TNF-α, eicosapentaenoic acid (EPA), which is a fatty acid having 20 carbon atoms, 4 double bonds, and n-3 series, was compared and examined.
Assay Method Human peripheral blood mononuclear cells (5 × 10 ⁵ / ml) and drug are pre-cultured in AIM-V medium (pH 7.4) for 30 minutes. Cells are stimulated by the addition of stimulants (concanavalin A; 20 μg / ml or lipopolysaccharide; 25 μg / ml) and cultured overnight at 37 ° C., 5% CO ₂ . The levels of various cytokines in the culture supernatant were measured by sandwich ELISA. The drug concentration was screened at 30, 10, 3, 1, 0.3 μM, and inhibition of production of various cytokines was evaluated by IC ₅₀ .
The 6,9,12,15-hexadecatetraenoic acid used in this example is the ethyl ester (purity 96%) produced by the method of Example 3.
The results are shown in Table 1. 6,9,12,15-hexadecatetraenoic acid (C16: 4) showed an inhibitory effect on any cytokine. Eicosapentaenoic acid was not effective in producing TNF-α even at a concentration of 30 μM.

上記の結果から、6,9,12,15-ヘキサデカテトラエン酸は、各種サイトカインの産生抑制効果を有することが示された。これらの結果は、6,9,12,15-ヘキサデカテトラエン酸に抗炎症作用、抗アレルギー作用、免疫抑制作用があることを示唆する。上記、サイトカインの中でも、インターロイキン−２とＴＮＦ-αは、特に各種疾患との関連が重要視されている因子である。

From the above results, it was shown that 6,9,12,15-hexadecatetraenoic acid has an effect of suppressing production of various cytokines. These results suggest that 6,9,12,15-hexadecatetraenoic acid has anti-inflammatory, anti-allergic and immunosuppressive effects. Among the cytokines described above, interleukin-2 and TNF-α are factors that are particularly emphasized in relation to various diseases.

Activity evaluation of 6,9,12,15-hexadecatetraenoic acid using an inflammation model
1. Inflammatory colitis model-I
A group of 5 male Wistar rats weighing 200 ± 10 g fasted for 24 hours. Peripheral colitis was induced by enteral administration of DNBS (2,4-Dinitrobenzensulfonic acid, 30 mg in 0.5 ml 30% ethanol). The compound (dissolved in 2% Polygly ester solution) was orally administered once daily for 7 days during the study period. DNBS was enterally administered 24 hours after the first drug administration and 2 hours after the second drug administration to induce colitis. The control group was administered DNBS at the same time. The other control group is a group not administered with DNBS. After continuous administration of the drug for 7 days, the large intestine was separated from the rat that was appropriately lethal at 24 hours, and the weight was measured. Diarrhea status was monitored throughout the study. When the abdominal cavity was opened, adhesion between the large intestine and other organs was observed, and ulcers of the large intestine were further evaluated. The ratio of the large intestine to body weight was calculated for each animal and quantified by the following formula.
(A) Large intestine weight per 100 g body weight = isolated large intestine x 100 / body weight on day 8
(B) Large intestine increase per 100 g body weight = (A)-Large intestine increase per 100 g body weight of untreated control
(C) Large intestine weight loss rate per 100g body weight = large increase in large intestine per 100g body weight of DNBS control-(B) / large increase in large intestine per 100g body weight of DNBS control In this study 6,9,12,15-hexa Decatetraenoic acid used was the ethyl ester (purity 78%) produced by the method of Example 3.

  The results are shown in Table 2. The colon weight per 100 g body weight of the untreated control group was 0.293 g, whereas when DNBS was administered, the colon weight increased to 0.784 g. This indicates that DNBS treatment caused inflammation in the large intestine. Administration of 6,9,12,15-hexadecatetraenoic acid (C16: 4 n-1) reduced the increase in colon weight to 0.656 g at 100 mg / kg oral administration. The decrease rate was 26%. In the case of oral administration at 300 mg / kg, the increase in the weight of the large intestine was reduced to 0.6 g. The decrease rate was 37%. As a positive control, the effect of Sulfasalazine was examined. Sulfasalazine reduced the increase in colon weight to 0.582 g by oral administration of 300 mg / kg. The decrease rate was 41%. As a result of the t-test, a significant difference was observed at P <0.05 between the 6,9,12,15-hexadecatetraenoic acid 100 mg and 300 mg groups and the control group administered with DNBS alone. It was. In addition, the Sulfasalazine 300 mg administration group showed a significant difference at P <0.01 compared with the control group to which DNBS alone was administered.

  Thus, 6,9,12,15-hexadecatetraenoic acid has almost the same effect as Sulfasalazine, a pharmaceutical agent that is actually used in clinical practice. Recognized in a flame model. This result indicates that 6,9,12,15-hexadecatetraenoic acid can be a therapeutic drug for Crohn's disease and ulcerative colitis, which are human diseases similar to this model.

2. Inflammatory colitis model-II
In a similar model, the effects of 6,9,12,15-hexadecatetraenoic acid and EPA were compared.
In this test, 6,9,12,15-hexadecatetraenoic acid is the ethyl ester produced by the method of Example 3 (purity 96%), EPA is triglyceride (lipid composition: triglyceride 99%, fatty acid composition: EPA 98% )It was used.
The results are shown in Table 3. The colon weight per 100 g body weight of the untreated control group was 0.225 g, whereas when DNBS was administered, the colon weight increased to 0.704 g. This indicates that DNBS treatment caused inflammation in the large intestine. Administration of 6,9,12,15-hexadecatetraenoic acid reduced the increase in colon weight to 0.574 g at 100 mg / kg oral administration. The decrease rate was 27%. It shows that the test results in the previous section were almost certainly reproduced. On the other hand, in the case of oral administration of EPA at 100 mg / kg, the increase in the weight of the large intestine was reduced to 0.607 g. The reduction rate was 20%. As a positive control, the effect of Sulfasalazine was examined. Sulfasalazine reduced the increase in large intestine weight to 0.574 g by oral administration of 300 mg / kg. The decrease rate was 35%. As a result of the t-test, a significant difference was observed at P <0.01 between the 100 mg administration group of 6,9,12,15-hexadecatetraenoic acid and the control group administered with DNBS alone. In addition, the Sulfasalazine 300 mg administration group showed a significant difference at P <0.01 compared with the control group to which DNBS alone was administered. There was no significant difference between the EPA-administered group and the control group that received DNBS alone at a risk rate of 5%.
From these results, not all fatty acids can be effective in such anti-inflammatory models as long as they are polyunsaturated fatty acids, in particular, 6,9,12,15-hexadeca This suggests that tetraenoic acid has a strong anti-inflammatory effect. EPA is also the first highly unsaturated fatty acid that has been widely studied to exhibit anti-inflammatory effects. This test can be considered as the first test result that has found that 6,9,12,15-hexadecatetraenoic acid may have a stronger anti-inflammatory effect than EPA.

3. Carrageenan edema model
A group of 6 male ICR mice with a body weight of 22 ± 2 g fasted for 24 hours. Carrageenan (50 μl of a 1% suspension) is injected into the paw 1 hour after oral administration of the drug. Using hind paw edema as an index of inflammation, measurement was performed with a dedicated measuring instrument 4 hours after carrageenan administration. This study is a very acute model that analyzes the effects of a single dose of 6,9,12,15-hexadecatetraenoic acid on edema formation. In this test, 6,9,12,15-hexadecatetraenoic acid was an ethyl ester (purity 78%) produced by the method of Example 3.
The results are shown in Table 4. In the control group, the volume of the hind paws increased to 5.7 (× 0.01 ml) due to edema caused by carrageenan administration. In the group in which 6,9,12,15-hexadecatetraenoic acid was administered 1 hour before carrageenan administration, the volume of the hind paws was suppressed to 4.5 (× 0.01 ml). With the positive control aspirin, the volume of the hind paws was kept at 3.3 (x0.01 ml). Thus, in an animal model, which is an acute inflammation model, 6,9,12,15-hexadecatetraenoic acid was shown to suppress inflammation even after a single administration.

4. Monoclonal antibody-induced collagenous arthritis A group of 5 BALB / cByJ mice weighing 20 ± 2 g. Four types of monoclonal antibodies against type II collagen (D8, F10, DI-2G, A2) were intravenously administered at 20 mg per mouse on day 0. On the third day, lipopolysaccharide (LPS, 25 μg / mouse) was intravenously administered to induce inflammation. The drug was orally administered once a day for 3 days from 1 hour after LPS administration on the third day. On the third day after LPS administration, hind limb edema was measured with a dedicated measuring instrument. In this test, 6,9,12,15-hexadecatetraenoic acid was an ethyl ester (purity 78%) produced by the method of Example 3.
The results are shown in Table 5. In the control group, the volume of the hind paws increased to 10 (x 0.01 ml) due to the administration of four monoclonal antibodies against type II collagen and the edema caused by the induction of LPS. In the group in which 6,9,12,15-hexadecatetraenoic acid was orally administered once a day for 3 days from 1 hour after LPS administration, the volume of the hind paws was suppressed to 5.6 (x 0.01 ml) It was. The decrease rate was 44%, and a significant difference was observed by t-test (P <0.05). Thus, in this model, 6,9,12,15-hexadecatetraenoic acid also caused inflammation. The inhibitory effect was shown.

5. Analgesic effect of 6,9,12,15-hexadecatetraenoic acid on rat beer yeast inflammation foot pain (Randall-Selitto method)
6,9,12,15-hexadecatetraenoic acid (using the ethyl ester produced by the method of Example 3 (purity 91.5%)) (specific gravity 0.9) was weighed into a 833 mg polypropylene centrifuge tube, and 3 A 25% polyglycerin ester aqueous solution was added and emulsified to prepare a test solution (30 mg / mL).
The test solution was administered by the oral route. Administration was performed once a day for 7 days. The dose of 6,9,12,15-hexadecatetraenoic acid was 300 mg / kg, and the dose volume was 10 mL / kg. The control group received the same volume of vehicle as the test solution. 1 g of brewer's yeast (Yeast, Brewers Bottom, manufactured by Sigma) was weighed, suspended in a physiological saline solution, and made up to 10 mL to prepare a 10 w / v% brewer's yeast physiological saline solution. One hour after the final administration of the test solution, inflammation was induced by subcutaneously administering 0.1 mL / mouse of 10 w / v% Brewers yeast physiological saline subcutaneously on the right hind footpad of the animal. At the 3 time points before the start of test solution administration, 1 hour before and 4 hours after the final administration of the test solution (3 hours after administration of 10 w / v% Brewers yeast saline solution), an analgesy meter (TK -201, Unicom), pressure stimulation was applied, and the pain threshold was measured.
The representative value of each group was expressed by the average value and standard error of the pain threshold. The equidispersity was tested by F test between the control group and the test solution group. Since it was equidispersion, Student's t test was performed. The number of animals used is 10 in each group.
The results are shown in Table 6. The average pain threshold (mean ± standard error) of the control group before the start of test solution administration, 1 hour before the last administration of the test substance and 3 hours after inflammation (4 hours after the last administration of the test substance) was 92 ± 1 mmHg, It was 90 ± 1 mmHg and 39 ± 3 mmHg, and the average pain response after 3 hours of inflammation decreased by 51 ± 3 mmHg.
The mean pain threshold before starting test solution administration of the 6,9,12,15-hexadecatetraenoic acid group, 1 hour before the last administration of the test substance and 3 hours after inflammation was 92 ± 1 mmHg, 89 ± 2 mmHg And 52 ± 3 mmHg, the average pain response at 3 hours after inflammation decreased by 37 ± 2 mmHg, significantly higher in the pain threshold at 3 hours after inflammation than the control group (p <0.01), significant in pain response A low value (p <0.01) was observed.

1. Distillation Distilled sardine oil ethyl ester (containing 1.9% 6,9,12,15-hexadecatetraenoic acid ethyl ester) as an ethyl ester using sodium ethylate under conditions of 135 ° C and 0.1 mmHg, A fraction containing 4.25% of 9,12,15-hexadecatetraenoic acid ethyl ester was obtained.
2. Urea addition 60.5 g of the obtained fraction was added dropwise to a 60 ° C methanol solution (600 ml) in which 180.0 g of urea was dissolved. After stirring for 1 hour, the mixture was further stirred for 16 hours at 4 ° C. And removed. The methanol in the obtained solution was distilled off, and then partitioned with 400 ml of hexane and 400 ml of distilled water, and the obtained hexane layer was further washed with 400 ml of distilled water three times. In addition, the aqueous layer obtained at the first time was extracted four times with 400 ml of hexane. Combine all hexane layers, dehydrate with anhydrous magnesium sulfate, filter anhydrous magnesium sulfate, distill off hexane, urea treated oil containing 17.1% of 6,9,12,15-hexadecatetraenoic acid ethyl ester 14.0 g was obtained. The recovery rate of 6,9,12,15-hexadecatetraenoic acid ethyl ester in urea addition treatment was 92.6%. The urea addition treatment under the same conditions was further performed 10 times to obtain a urea-treated oil containing 17.4% of 6,9,12,15-hexadecatetraenoic acid ethyl ester.
3. Column An open column filled with octadecylsilylated silica gel (100-200mesh manufactured by Fuji Silysia Chemical Co., Ltd.) with 35 g of urea-treated oil of 17.4% of the obtained 6,9,12,15-hexadecatetraenoic acid ethyl ester (7.5 cmφ × 25 cm), and after passing 800 ml of 95% methanol, 200 ml fractions were collected. As a result of distilling off the solvent of the obtained fraction, 79.7% of 6,9,12,15-hexadecatetraenoic acid ethyl ester 1.3 g (recovery rate 17.5%) was obtained from fraction 3. In the other fractions, 5.3 g of 6,9,12,15-hexadecatetraenoic acid ethyl ester (recovery rate 51.6%) was 59.5% from fraction 4, and 66.4,6,12,15-hexa was 26.4% from fraction 5. 6.0 g of decatetraenoic acid ethyl ester (recovery rate 26.2%) was obtained, and the total recovery rate was 95.3%.
Column treatment under the same conditions was repeated several times, and fractions with low purity were collected to give 31.2% 6,9,12,15-hexadecatetraenoic acid ethyl ester. 94.7% of 6,9,12,15-hexadecatetraenoic acid ethyl ester 2.3 g (recovery 21.1%) was obtained. In the other fractions, 10.1 g (recovery 56.3%) of 6,9,12,15-hexadecatetraenoic acid ethyl ester 58.2% from fraction 4 and 26.3% 6,9,12,15-hexa from fraction 5 Decatetraenoic acid ethyl ester 8.7 g (recovery rate 21.9%) was obtained, and the total recovery rate was 99.2%.

As a result of analyzing 96.0% of 6,9,12,15-hexadecatetraenoic acid ethyl ester prepared in the same manner as described above by 400 MHz 1H-NMR, GC-MS, 6,9, It was confirmed that it was 12,15-hexadecatetraenoic acid.
1H-NMR (400MHz, CDCl3): 1.25 (t, J = 7.1Hz, 3H), 1.38 (m, 2H), 1.6 (m, 2H), 2.1 (m, 2H), 2.30 (t, 2H), 2.8 (m, 6H), 4.12 (quart., J = 7.1Hz, 2H), 5.0 (m, 2H), 5.3-5.4 (m, 6H), 5.7 (m, 1H)
Mass m / z: 276 (M +.), 261, 247, 235, 208, 189, 171, 161.

Manufacturing method using high-performance liquid chromatograph A high-performance liquid chromatograph was used. 200 μl of 50% methanol solution (about 90 mg as oil) of 6,9,12,15-hexadecatetraenoic acid ethyl ester (34.5%) was added to Cosmo Seal 5C18-AR packed column (20.0 mm I.D. × by Nacalai Tesque) 250mm) was collected using a high performance liquid chromatograph (solvent methanol, flow rate 5ml / h), 95.4% 6,9,12,15-hexadecatetraenoic acid ethyl ester 18.7mg (recovery rate) 57.3%).
200 μl of a 94.5% 50% methanol solution of 6,9,12,15-hexadecatetraenoic acid ethyl ester (approximately 90 mg as an oil) obtained by repeated fractionation using a high performance liquid chromatograph under the same conditions When fractionated again under the same conditions, 99.1% 6,9,12,15-hexadecatetraenoic acid ethyl ester 15.9 mg (recovery 18.5%), 97.5% 6,9,12,15-hexa Decatetraenoic acid ethyl ester 28.1 mg (recovery rate 32.2%) was obtained.

Prophylactic or therapeutic agents for inflammatory diseases or diseases caused by inflammatory diseases such as colitis and arthritis can be provided. Moreover, since it is a fatty acid derived from food and high safety can be expected, a functional food having a function of preventing or treating inflammatory diseases can be provided.

Claims (7)

An agent for preventing or treating inflammatory diseases comprising 6,9,12,15-hexadecatetraenoic acid or a pharmaceutically acceptable salt or ester thereof as an active ingredient. The pharmaceutically acceptable salt of 6,9,12,15-hexadecatetraenoic acid is any one of sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, pyridine salt, and triethylamine salt. 1. An agent for preventing or treating inflammatory diseases. A triglyceride, diglyceride or monoglyceride in which the pharmaceutically acceptable ester of 6,9,12,15-hexadecatetraenoic acid contains 6,9,12,15-hexadecatetraenoic acid as a constituent fatty acid, or The preventive or therapeutic agent for inflammatory diseases according to claim 1, which is a lower alcohol ester of 6,9,12,15-hexadecatetraenoic acid.   The inflammatory disease preventive or therapeutic agent according to claim 1, 2 or 3, wherein the inflammatory disease is a chronic inflammatory disease.   A kit for the prophylaxis or treatment of inflammatory diseases comprising the inflammatory disease therapeutic agent according to any one of claims 1 to 4 and instructions for use. 6,9,12,15 in the constituent fatty acids using fish oil as raw material by any method selected from distillation, urea addition, column chromatography, enzyme reaction, supercritical fluid extraction, silver complex formation, low temperature fractionation, solvent fractionation -Functional food containing an amount for ingesting 50 to 1000 mg of triglyceride enriched in hexadecatetraenoic acid content or ethyl ester of 6,9,12,15- hexadecatetraenoic acid per day. Capsules containing functional foods containing triglycerides enriched in 6,9,12,15-hexadecatetraenoic acid content in constituent fatty acids or ethyl ester of 6,9,12,15-hexadecatetraenoic acid The functional food according to claim 6.